In this thesis we further explored the immune responses elicited by the HIV-DNA and HIV-MVA vaccine
regimen and the effect of boosting with a third HIV-MVA or an envelope protein.

In study I, we evaluated the HIV vaccine-induced antibody responses in sera collected from 29 HIVIS03
vaccinees at baseline and four weeks after the second HIV-MVA. High titers of neutralizing antibodies (NAbs)
(median 357) were detected using an infectious molecular clone (IMC)/peripheral blood mononuclear cell
(PBMC) assay. The NAbs were significantly (but not completely) removed upon depletion of natural killer (NK)
cells from PBMC (p=0.0039), indicating a role for Fc–receptor mediated antibody function. ADCC-mediating
antibodies were demonstrated in the majority (97%) of the vaccinees against CRF01_AE and/or subtype B. The
magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with NAbs
against CM235 in the IMC/PBMC assay.

In studies II and III we explored the duration of immune responses in individuals primed with HIV-DNA and
boosted with HIV-MVA in the HIVIS03 trial and the effect of a late third HIV-MVA. Twenty volunteers who
had previously received three HIV-DNA and two HIV-MVA immunizations in the HIVIS03 trial were given a
third HIV-MVA, three years after the second HIV-MVA boost (HIVIS06). A high proportion of vaccinees
showed durable binding antibodies, 90% to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B
gp160 (median titer 100) three years after the second HIV-MVA. The majority of vaccinees had detectable
ADCC–mediating antibodies, 70% against CRF01_AE virus–infected cells (median titer 239) and 84% against
CRF01_AE gp120–coated cells (median titer 499). Furthermore, 74% of vaccinees still had IFN- γ ELISpot
responses, 63% to Gag and 42% to Env. After the third HIV-MVA, there was an increase in Env-binding
antibodies and ADCC-mediating antibodies relative to the response seen at the 3-years time point. The frequency
of IFN- γ ELISpot responses increased to 95% against Gag or Env, and 90% to both Gag and Env, p = 0.064 and
p = 0.002, respectively, after the third HIV-MVA. All 19 (100%) evaluable vaccinees had IgG antibodies to
V1V2 CRF01_AE A244 after the second HIV-MVA with a median titer of 3200. A high proportion (75%) of
the vaccinees still had V1V2 IgG antibodies to CRF01_AE A244 three years after the second HIV-MVA, which
increased to 95% after the third HIV-MVA. The magnitude of response before and after the third MVA
increased significantly from a median titer of 400 to 1600, p<0.0001, but not to the same level as after the second
HIV-MVA (p=0.025). Surface plasmon resonance/Biacore analysis data supported the ELISA findings. V1V2-
specific IgG1 antibody responses were more frequently detected than V1V2-specific IgG3 antibodies. AntiV1V2 IgG1 responses decreased after three years but could be boosted by the third HIV-MVA in the majority of
the vaccinees.

In study IV, we evaluated the safety and impact of boosting with subtype C CN54rgp140 Env protein adjuvanted
in glucopyranosyl lipid A-aqueous formulation (GLA-AF) in volunteers previously given three HIV-DNA,
followed by two HIV-MVA in the TaMoVac 01 trial. Forty volunteers (35 vaccinees and five placebo recipients)
were given two CN54rgp140/ GLA-AF immunizations 30–71 weeks after the second HIV-MVA. The vaccine
was safe and well tolerated, except for one incident of asymptomatic hypoglycemia. After the second HIV-MVA
vaccination, 34 (97%) of the vaccinees developed Env-specific binding antibodies, whereas 79% and 84%
exhibited IFN- γ ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C, B and
CRF01_AE Env were significantly boosted by the CN54rgp140/GLA-AF immunizations, while functional
antibodies were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen
and IFN- γ ELISpot responses to Env peptides were significantly enhanced.

In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune
responses with remarkable durability, and a third HIV-MVA significantly boosted both antibody and cellular
immune responses. Binding antibody responses and Env-specific cell-mediated immune responses but not
functional antibody responses, increased after boosting with two CN54rgp140/GLA-AF immunizations
following priming with HIV-DNA and HIV-MVA.